Compositions and methods for treating immune disorders

ABSTRACT

Green tea polyphenol compositions and methods of their use are provided. Certain aspects provide methods for modulating expression of one or more autoantigens using the disclosed green tea polyphenol compositions. Representative green tea polyphenols include, but are not limited to (-)-epigallocatechin-3-gallate. Other aspects provide methods for treating autoimmune disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of copending application Ser. No.11/916,832, entitled “Compositions and Methods for Treating ImmuneDisorders,” by Stephen D. Hsu, et al., which is a filing under 35 U.S.C.§371 of PCT/US2006/022554 filed with the U.S. Receiving Office of thePatent Cooperation Treaty on Jun. 9, 2006, which claims benefit of andpriority to U.S. Provisional Application No. 60/689,747, filed on Jun.10, 2005, all of which are hereby incorporated by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Agreement R21CA097258-01 A1 awarded by the National Institutes of Health. The U.S.Government has certain rights in the invention.

TECHNICAL FIELD

Aspects of the disclosure are generally related to compositions andmethods for modulating autoantigen expression, more particularly togreen tea polyphenol compositions and methods of their use, for examplein the treatment or prophylaxis of autoimmune disorders.

RELATED ART

The prevalence of autoimmune disorders in the United States is estimateat more than 8.5 million. Autoimmune reactions can cause inflammationand apoptosis of target cells, leading to destruction of multipletissues and organs. Patients with autoimmune diseases developautoantibodies against a diverse group of macromolecules involved innormal functions. For example, anti-SS-A/Ro and anti-SS-B/Laautoantibodies are primary markers for certain autoimmune diseases suchas lupus erythematosus and Sjögren's syndrome. Existing treatments ofautoimmune diseases have focused on the immune system, not theautoantigens that could trigger or sustain a positive feedback loop ofinflammation and apoptosis.

Lupus Autoimmune Disorders

Lupus is one of more than 60 types of autoimmune disorders, and is oneof the most destructive. Clinical classification of lupus includessystematic lupus erythematosus (SLE), discoid lupus erythematosus (DLE),subacute cutaneous lupus (SCLE), drug induced lupus, and neonatal lupus.SLE is the most prevalent form and may affect, multiple tissues such asjoints, skin, kidneys, heart, lungs, blood vessels, and brain. Itaffects mostly young females of childbearing age. Lupus can cause severejoint and muscle pain, extreme exhaustion, fevers, and skin rashes, andcan lead to organ failure, scars and death. The skin manifestations ofSLE arid DLE are slightly different. DLE affects mainly the skin and theoral cavity with disk-shaped lesions while SLE affects multiple organs.Skin manifestations occur in about 25% of SLE patients, with butterflyshaped lesions distributed on the face and ears. It is believed thatcutaneous LE affects 14.6 to 68 per 100 000 people (Callen, J. P. (2004)Br J Dematol. 151 (4):731-6).

Sjögren 's Syndrome

Sjögren's syndrome (SS) is another autoimmune disorder that affectsmultiple tissues. Primary SS is associated with lymphocyticinfiltrations of the salivary and lacrimal glands and eventual atrophy,leading to a loss of fluid production. The salivary component of SS isdefined as xerostomia, with symptoms generally referred to as salivaryhypofunction (Daniels, T. E. and Fox, R. I. (1992). Rheum Dis Clin NorthAm. 18 (3):571-89). If not treated, xerostomia may lead to oralcomplications (Daniels T. E. and Wu, A J. (2000) Calif Dent Assoc. 28(12):933-41). Estimates of the prevalence of SS are affected by thecriteria used for diagnosis. However, genuine differences betweenvarious regions and communities exist (Fox, R. I. (1997) Clin Lab Med.17 (3):43 1-44; Vitali, C. et al. (2002) Ann Rheum Dis. 61 (6):554-8).The world-wide distribution is believed to be 1/2500 (Kang, H. (1993)).In the United States, SS affects approximately 1% of the population(Carsons, S. (2001) Am J Manag Care. 7 (14 Suppl):5433-43.24). In China,one regional study with 26,000 subjects suggested the prevalence ofprimary SS was only 0.03% (Zhang, N. (1995) Chin Med J (Engl). 108(10):787-8). In Japan, the estimated crude prevalence rates for SS wereonly 1.9 and 25.6 per 100,000 population in males and females,respectively (Yoshida, S. (1999) Nippon Rinsho. 57 (2):360-3). A surveyconducted by the Japanese Ministry of Health and Welfare indicated theSS prevalence was just 0.06% among females (Miyasaka, N. (1995) NipponRinsho. 53 (10):2367-70).

As for xerostomia, one study showed that among a group of 1003 Japaneseindividuals with an average age of 66, about 9.1% experienced dry mouthduring eating (Ikebe, K. et al. (2001) Spec Care Dentist. 21 (2):52-9),whereas in the United States, one epidemiological study found that in agroup of 2481 individuals aged 65-84 years old, 27% reported either drymouth or dry eyes (Schein, O. D. et al. (1999) Arch Intern Med. 159(12): 1359-63), and another found that dry mouth ranged from 10% amongpersons over age 50 to 40% for persons over age 65 (Billings R J., etal. (1996) Community Dent Oral Epidemiol. (5):3 12-6). Although precisestatistical comparison between the U.S. population and either theJapanese or Chinese population is not available, it is apparent that SSand xerostomia are more prevalent in the U.S. population, particularlyamongst the elderly.

SS is not a curable or preventable disease at present, and whether itcan be prevented or delayed is unknown. Treatment is generallysymptomatic and supportive. For xerostomia and xerophthalmia, artificiallubricants are commonly used as saliva or tear substitutes (Baudouin, C.et al. (2004) Rev Med Interne. 25 (5):376-82). In recent years, salivarystimulants, such as pilocarpine and cevimeline, have been approved bythe FDA to treat xerostomia (Fox, R. I. (2003) Expert Opin lnvestigDrugs. 12 (2):247-54); Cassolato, S. F. and Turnbull, R. S. (2003)Gerodontology. 20 (2):64-77; Porter, S. R. et al. (2004) Oral Surg OralMed Oral Pathol Oral Radiol Endod. 97 (1):28-46). In addition, oraladministration of interferon γ (IFN-γ) was effective in improving salivaproduction in patients with primary SS (Khurshudian, A. V. (2003) OralSurg Oral Med Oral Pathol Oral Radiol Endod. 95 (1):38-44). However,long-term adverse effects have not been evaluated for these therapies.

Thus, there is a need for additional compositions and methods forpreventing and treating autoimmune diseases or symptoms associated withsuch diseases.

SUMMARY

Aspects of the disclosure generally provide green tea polyphenolcompositions and methods of their use, for example in decreasingautoantigen expression in a host or cell. In particular, it has beendiscovered that (-)-epigallocatechin-3-gallate modulates expression ofautoantigens. Downregulation of autoantigens using the disclosed greentea polyphenol compositions can be used to treat autoimmune diseases orsymptoms associated with autoimmune diseases. Increasing expression ofautoantigens can be used to assist in the purification and isolation ofautoantigens, for example to generate antibodies that can be used asdiagnostics.

One aspect of the disclosure provides a method for decreasingautoantigen expression in a cell by contacting the cell with acomposition having one or more green tea polyphenols. The cell contactedwith the one or more green tea polyphenols shows a decreased level ofautoantigen expression relative to a control. In certain aspects, thegreen tea polyphenol is (-)-epigallocatechin-3-gallate, apharmaceutically acceptable salt or prodrug thereof. Representativeautoantigens include those listed in Table 1, for example SS-A, SS-B,fodrin, centromere protein, golgin-67, coilin, and PARP. In otheraspects, the composition further includes one or more green teapolyphenols such as (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin-3-gallate, proanthocyanidins, enantiomers thereof, epimersthereof, isomers thereof, combinations thereof, and prodrugs thereof. Itwill be appreciated that the disclosed methods and compositions can beused to modulate, in particular reduce the expression of at least twoautoantigens relative to a control.

Another aspect provides a method for modulating autoantigen geneexpression by administering to a host one or more green tea polyphenolsin an amount effective to reduce or increase expression of anautoantigen gene compared to a control. Reduced expression of theautoantigen gene can occur at the transcriptional or translationallevel.

Still another aspect provides a method for treating an autoimmunedisease by administering to a host one or more green tea polyphenols inan amount effective to decrease expression of one or more autoantigens.Representative autoimmune diseases include, but are not limited toHashimoto's thyroiditis, pernicious anemia, Addison's disease, type Idiabetes, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiplesclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease,scleroderma, psoriasis, xerostomia, and xeropthalmia.

Another aspect provides a method for treating xerostomia byadministering to a host one or more green tea polyphenols in an amounteffective to decrease expression of one or more autoantigens, whereinthe decreased expression of the one or more autoantigens occurs in oneor more salivary gland cells of the host.

Yet another aspect provides a method for treating xerophthalmia byadministering to a host one or more green tea polyphenols in an amounteffective to decrease expression of one or more autoantigens in one ormore of the host's lacrimal gland cells.

Still another aspect provides a method for treating psoriasis byadministering to a host one or more green tea polyphenols in an amounteffective to decrease expression of one or more autoantigens in one ormore epidermal cells of the host.

Another aspect provides a use of a green tea polyphenol in themanufacture of a medicament for the treatment of an autoimmune disease,in particular, psoriasis, xerostomia, or xerophthalmia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a gel indicating mRNA levels of SS-B/La and SS-A/Ro innormal, immortalized, and tumor cells treated with(-)-epigallocatechin-3-gallate.

FIG. 2 shows Western blot results of autoantigen protein levels in(-)-epigallocatechin-3-gallate-treated NHEK and NS-SV-Ac cells.

FIG. 3 shows mice treated with green tea polyphenols have reducedautoantibodies.

FIG. 4A shows submandibular gland sections from control NOD mice.

FIG. 4B shows submandibular gland sections from NOD mice fed with greentea polyphenols.

FIG. 5 shows a bar graph of the densities of the average focal areas inNOD mice fed green tea polyphenols compared to control mice.

FIG. 6 shows a bar graph of cell viability indicating that ECGC protectscells against TNF-induced cytotoxicity.

FIG. 7A shows a bar graph of cell viability indicating that inhibitionof p38 abolishes EGCG protection.

FIG. 7B shows a bar graph of cell viability indicating that inhibitionof MEK abolishes the EGCG effect.

FIG. 8 shows an autoradiograph indicating that p38 is rapidly andspecifically phosphorylated within 30 min in acinar cell-derivedNS-SV-AC cells contacted with EGCG.

FIG. 9 shows micrographs indicating local lymphocyte infiltration in thesubmandibular glands of early GTP-treated MRL and NOD mice.

DETAILED DESCRIPTION 1. Definitions

Before explaining the various embodiments of the disclosure, it is to beunderstood that the invention is not limited in its application to thedetails of construction and the arrangement of the components set forthin the following description. Other embodiments can be practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced. Where permissible, the disclosuresof these publications, patents and published patent specifications arehereby incorporated by reference in their entirety into the presentdisclosure to more fully describe the state of the art.

To facilitate understanding of the disclosure, the following definitionsare provided:

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a factor” refers to one or mixtures of factors,and reference to “the method of treatment” includes reference toequivalent steps and methods known to those skilled in the art, and soforth.

The term “autoantigen” refers to an antigen produced by an organism andrecognized by the organism's immune system. Representative autoantigensinclude, but are not limited to those listed in Table 1.

The term “autoimmune disease or disorder” refers to conditions caused byan immune response against the body's own tissues or cells.Representative autoimmune disorders include, but are not limited toHashimoto's thyroiditis, pernicious anemia, Addison's disease, type Idiabetes, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiplesclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease,scleroderma, psoriasis, xerostomia, and xeropthalmia.

The term “cell” refers to a membrane-bound biological unit capable ofreplication or division.

The term “Green Tea Polyphenols or GTP” refers to polyphenolic compoundspresent in the leaves of Camellia sinensis. Green tea polyphenolsinclude, but are not limited to (-)-epicatechin (EC),(-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECF),(-)-epigallocatechin-3-gallate (ECGC), proanthocyanidins, enantiomersthereof, epimers thereof, isomers thereof, combinations thereof, andprodrugs thereof.

The term “host” refers to a living organism, including but not limitedto a mammal such as a primate, and in particular a human.

The term “isolated,” when used to describe the various compositionsdisclosed herein, means a substance that has been identified andseparated and/or recovered from a component of its natural environment.For example an isolated polypeptide or polynucleotide is free ofassociation with at least one component with which it is naturallyassociated. Contaminant components of its natural environment arematerials that would typically interfere with diagnostic or therapeuticuses for the polypeptide or polynucleotide and may include enzymes, andother proteinaceous or non-proteinaceous solutes. An isolated substanceincludes the substance in situ within recombinant cells. Ordinarily,however, an isolated substance will be prepared by at least onepurification step.

The term “operably linked” refers to a juxtaposition wherein thecomponents are configured so as to perform their usual function. Forexample, control sequences or promoters operably linked to a codingsequence are capable of effecting the expression of the coding sequence,and an organelle localization sequence operably linked to protein willdirect the linked protein to be localized at the specific organelle.

The term “pharmaceutically” acceptable carrier” refers to a carrier ordiluent that does not cause significant irritation to an organism anddoes not abrogate the biological activity and properties of theadministered compound.

The term “pharmaceutically acceptable salt” refers to those salts whichretain the biological effectiveness and properties of the free bases andwhich are obtained by reaction with inorganic or organic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid,succinic acid, tartaric acid, citric acid, and the like.

A “pharmaceutical composition” refers to a mixture of one or more of thegreen tea polyphenols described herein, or a pharmaceutically acceptablesalts thereof, with other chemical components, such as physiologicallyacceptable carriers and excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to anorganism.

The term “prodrug” refers to an agent, including nucleic acids andproteins, which is converted into a biologically active form in vivo.Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent compound. They may, for instance,be bioavailable by oral administration whereas the parent compound isnot. The prodrug may also have improved solubility in pharmaceuticalcompositions over the parent drug. A prodrug may be converted into theparent drug by various mechanisms, including enzymatic processes andmetabolic hydrolysis. Harper, N.J. (1962). Drug Latentiation in Jucker,ed. Progress in Drug Research, 4:221-294; Morozowich et al. (1977).Application of Physical Organic Principles to Prodrug Design in E. B.Roche ed. Design of Biopharmaceutical Properties through Prodrugs andAnalogs, APhA; Acad. Pharm. Sci.; E. B. Roche, ed. (1977). BioreversibleCarriers in Drug in Drug Design, Theory and Application, p APhA; H.Bundgaard, ed. (1985) Design of Prodrugs, Elsevier; Wang et al. (1999)Prodrug approaches to the improved delivery of peptide drug, Curr.Pharm. Design. 5 (4):265-287; Pauletti et al. (1997). Improvement inpeptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv.Drug. Delivery Rev. 27:235-256; Mizen et al. (1998). The Use of Estersas Prodrugs for Oral Delivery of [beta]-Lactam antibiotics, Pharm.Biotech. 11:345-365; Gaignault et al. (1996). Designing Prodrugs andBioprecursors I. Carrier Prodrugs, Pract. Med. Chem. 671-696; M.Asgharnejad (2000). Improving Oral Drug Transport Via Prodrugs, in G. L.Amidon, P. I. Lee and E. M. Topp, Eds., Transport Processes inPharmaceutical Systems, Marcell Dekker, p. 185-218; Balant et al. (1990)Prodrugs for the improvement of drug absorption via different routes ofadministration, Eur. J. Drug Metab. Pharmacokinet, 15 (2): 143-53;Balimane and Sinko (1999). Involvement of multiple transporters in theoral absorption of nucleoside analogues, Adv. Drug Delivery Rev., 39 (1-3): 183-209; Browne (1997). Fosphenytoin (Cerebyx), Clin.Neuropharmacol. 20 (1): 1-12; Bundgaard (1979). Bioreversiblederivatization of drugs—principle and applicability to improve thetherapeutic effects of drugs, Arch. Pharm. Chemi. 86 (1): 1-39; H.Bundgaard, ed. (1985) Design of Prodrugs, New York: Elsevier; Fleisheret al. (1996). Improved oral drug delivery: solubility limitationsovercome by the use of prodrugs, Adv. Drug Delivery Rev. 19 (2):115-130; Fleisher et al. (1985). Design of prodrugs for improvedgastrointestinal absorption by intestinal enzyme targeting, MethodsEnzymol. 112: 360-81; Farquhar D, et al. (1983). Biologically ReversiblePhosphate-Protective Groups, J. Pharm. Sci., 72 (3): 324-325; Han, H. K.et al. (2000). Targeted prodrug design to optimize drug delivery, AAPSPharmSci., 2 (1): E6; Sadzuka Y. (2000). Effective prodrug liposome andconversion to active metabolite, Curr Drug Metab., 1 (1):31-48; D. M.Lambert (2000) Rationale and applications of lipids as prodrug carriers,Eur. J. Pharm. Sci., 11 Suppl 2:S15-27; Wang, W. et al. (1999) Prodrugapproaches to the improved delivery of peptide drugs. Curr. Pharm. Des.,5 (4):265-87.

The term “treating or treatment” refers to alleviating, reducing, orinhibiting one or more symptoms or physiological aspects of a disease,disorder, syndrome, or condition.

2. Green Tea Compositions

One embodiment provides a composition having one or more green teapolyphenols, in particular (-)-epigallocatechin-3-gallate, apharmaceutically acceptable salt, prodrug, or derivative thereof, in anamount effective to modulate expression of one or more autoantigens in ahost compared to a control. Modulate means to increase, decrease,reduce, or inhibit expression of an autoantigen in a host or cell.Experimental controls or control groups are known in the art. Generally,the effect of the green tea polyphenol composition on thedownregulation, inhibition, or modulation of an autoantigen can becompared to the effect of the composition without the green teapolyphenol on the down regulation, inhibition, or modulation of anautoantigen. Representative hosts include mammals such as humans.

A derivative or variant of a green tea polyphenol includes green teapolyphenols having chemical modifications to increase solubility orbioavailability in a host. These chemical modifications include theaddition of chemical groups having a charge under physiologicalconditions as well as the conjugation of the green tea polyphenol toother biological moieties such as polypeptides, carbohydrates, lipids,or a combination thereof.

The disclosed green tea polyphenol composition can decrease or inhibitthe expression of an autoantigen in any cell expressing an autoantigenor capable of expressing an autoantigen. Representative cells include,but are not limited to a primary epidermal keratinocyte, a salivarygland cell, or a lacrimal gland cell.

Autoantigens include, but are not limited to anti-nuclear autoantibodiessuch as SS-A/Ro, SS-B/La, centromere protein (CNEP) A, B, C, dsDNA,polymyositis-scleroderma (PM-scl), RNA polymerases, poly(ADP)ribosepolymerase (PARP), uridine rich 1 small nuclear ribonucleoprotein (U1snRNP), Smith antigen (Sm), ribosomal-P, histidyl t-RNA synthase (Jo-1),and DNA topoisomerase 1 (Scl-70) as well as those listed in Table 1.

Another embodiment provides a pharmaceutical composition including oneor more green tea polyphenols in combination with a pharmaceuticallyacceptable carrier, diluent, or excipient. The one or more green teapolyphenols are in an amount modulate the expression of an autoantigenin a host. In some embodiments, the one or more green tea polyphenolsare in an amount effective to inhibit, reduce, or decrease theexpression of two or more autoantigens in a host. In other embodiments,the active ingredient in the composition consists essentially of(-)-e[rho]igallocatechin-3-gallate, a pharmaceutically acceptable saltor prodrug thereof. The active ingredient can be in the form a singleoptical isomer. Typically, one optical isomer will be present in greaterthan 85%, 90%, 95%, or 99% by weight compared to the other opticalisomer. It will be appreciated that the composition can also include atleast one additional active ingredient, for example a secondtherapeutic. Additional description of the disclosed pharmaceuticalcompositions is provided below.

3. Methods of Use

One embodiment provides a method for modulating expression of one ormore autoantigens in a cell or host by contacting the cell or host witha green tea polyphenol composition. The green tea polyphenol compositionincludes an amount of one or more green tea polyphenols,pharmaceutically acceptable salts, prodrugs, or derivatives thereof inan amount effective to modulate the expression of an autoantigen. Theexpression of the autoantigen can be increased or decreased as comparedto a control. The modulation of autoantigen expression can be at thetranscriptional or translational stage. For example, the amount of mRNAencoding one or more autoantigens can be reduced or increased in cellscontacted with a green tea polyphenol composition relative to a control.Alternatively, the amount of protein corresponding to an autoantigen canbe increased or reduced in cells contacted with the disclosed green teapolyphenol compositions.

Another embodiment provides a method for treating an autoimmune diseaseby administering to a host an amount of one or more green teapolyphenols effective to reduce, inhibit, or decrease the expression ofan autoantigen relative to a control. Representative autoimmune diseasesinclude, but are not limited to Hashimoto's thyroiditis, perniciousanemia, Addison's disease, type I diabetes, rheumatoid arthritis,systemic lupus erythematosus (SLE), dermatomyositis, Sjogren's syndrome(SS), lupus erythematosus, multiple sclerosis, myasthenia gravis,Reiter's syndrome, Grave's disease, scleroderma, psoriasis, xerostomia,and xeropthlmia. Both SLE and SS are characterized by the production ofautoantibodies that have been implicated in the pathogenic effects ontissues. To date, a large number of autoantigens have been identified inSLE. Sera from lupus patients often have high titers of anti-nuclearautoantibodies (ANAs) that target components of the nucleus (Sawalha andHarley, (2004) Curr Opin Rheumatol. 16 (5):534-40). These ANAs includeSS-A/Ro, SS-B/La, centromere protein (CNEP) A, B, C, dsDNA,polymyositis-scleroderma (PM-scl), RNA polymerases, [rho]oly(ADP)ribosepolymerase (PARP), uridine rich 1 small nuclear ribonucleoprotein (U1snRNP), Smith antigen (Sm), ribosomal-P, histidyl t-RNA synthase (Jo-1),and DNA topoisomerase 1 (Scl-70) (Reeves, G. E. (2004) Intern Med J. 34(6):338-7). ANAs are also found in about 70% of patients with SS, andautoantibodies against SS-A/Ro and SS-B/La are found in about 95% and87% of primary SS patients, respectively (Rehman H. U. (2003) Yonsei MedJ. 44 (6):947-54). Elevated levels of SS-A/Ro and SS B/La mRNA werefound in the salivary tissues of SS patients with xerostomia (Bolstad A.I., et al. (2003) Arthritis Rheum 48:174-85). Lupus-associatedautoantigens also include golgins present in the Golgi apparatus andcoilin proteins (Stinton, L. M. et al. (2004) Clin Immunol. 110(1):30-44).

The mechanism leading to presentation of autoantigens to the immunesystem is unknown. One mechanism that may initiate the autoimmuneresponse is the translocation of nuclear autoantigens onto the cellmembrane during apoptosis, where they are exposed to antigen-presentingcells such as macrophages and dendritic cells (Manganelli and Fietta,2003). During apoptosis, autoantigens redistribute to form apoptoticbodies and blebs, where autoantigens such as SS-A/Ro, SS-B/La, Ku,poly(ADP)ribose polymerase (PARIP), fodrin, Golgins and nuclear mitoticapparatus protein (NuMA) are clustered as subcellular structures. Anaberrant structure of these autoantigen complexes may contribute to theautoimmune response (Rosen and Casciola-Rosen, 2004). B cells can bestimulated to proliferate and produce autoantibodies by perturbations inthe levels of cytokines. Although the exact role of autoantibodies inthe pathogenesis SLE or SS remains unclear, it is thought they areinvolved directly in some of the clinical manifestations (Mamula et al.,1994).

Another embodiment provides a method for treating an autoimmune diseaseby administering to a host an amount of green tea polyphenol, forexample ECGC, effective to down-regulate the expression of autoantigensat the mRNA and/or protein levels, for example in different epithelialcell types. Still another embodiment provides a method for treating anautoimmune disease or disorder by administering to a host an amount ofgreen tea polyphenol, for example ECGC, effective to reduce or inhibitexpression of an autoantigen, reduce or inhibit apoptosis; and reduce orinhibit inflammation. The reduction in expression of an autoantigen,reduction of apoptosis, and the reduction of inflammation can be in anycell capable of expressing an autoantigen, for example in epithelialcells such as salivary gland cells, lacrimal gland cells, or primaryepithelial keratinocyte.

Results from the Affymetrix gene expression analyses in Example 1 andTable 1 indicated that EGCG modulated the expression of a group of majorautoantigens in NHEK, with several genes showing a 2-fold or morereduction in mRNA levels, in some cases after an initial increase. Thevarious different patterns in the kinetics of change among differentautoantigens suggests that different regulator mechanisms could beinvolved. (McArthur et al., 2002). Expression of SS-A/Ro 52 (which wasnot represented on the Affymatrix array) was shown to be reduced by EGCGat the mRNA and protein level in two different epithelial cell linesusing RT-PCR and Western analyses. Similarly, microarray, RT-PCR andWestern analyses demonstrated that EGCG reduced expression of SS-B/La.In contrast, the microarray analysis showed that SS-A/Ro 60 mRNA levelswere not significantly altered by EGCG. Interestingly, oxidative stressinduces cell surface expression of SS-A/Ro 52, but not SS-A/Ro 60autoantigen on NHEK cells (Saegusa et al., 2002). Since green teapolyphenols inhibit the effects of oxidative stress on normal cells,this may be one mechanism by which EGCG reduces expression ofautoantigens (Yamamotu et al., 2004). An inhibitory effect of EGCG onprotein levels of four other autoantigens was demonstrated by Westernanalysis in NHEK and NS-SV-AC cells. The kinetics of reduction inprotein levels differed somewhat between the autoantigens. This couldreflect regulation via different pathways, or differences in mRNA orprotein stability, or in protein trafficking

Another embodiment provides a method for reducing or inhibitingSS-induced salivary gland destruction by administering to a hostexpressing one more symptoms of SS an amount of green tea polyphenolseffective to reduce or inhibit one or more of apoptosis, autoantigengene expression, or cytokine production. Examples 4 and 5 show asignificant reduction of serum total autoantibody levels in GTP-treatedNOD animals, compared with the untreated control NOD animals (FIG. 3).The NOD mouse is a known model for SS. Similarly, the size oflymphocytic infiltrate foci was also reduced after GTP treatment (FIGS.4 and 5). Thus, another embodiment provides a method for reducinglymphocytic infiltration of salivary glands by administering to a hostan amount of green tea polyphenols, for example ECGC, effective toreduce or inhibit the expression of an autoantigen.

The disclosed in vivo evidence indicates that GTPs have a beneficialeffect against autoimmune responses in the NOD mouse. Green teaconsumption by humans leads to an increase of secreted salivary GTPs, ina concentration range (50+μM) 10 times higher than the serum levels(Yang et al., 1999). Oral exposure to GTPs in this mouse model alsoresults in elevation of salivary GTPs to protective concentrations. TheGTP-treated group also had an average one week delay in the onset ofautoimmune diabetes, and while all of the 15 GTP-fed NOD mice survivedthe 3-week disease progression period, two untreated control mice diedduring this period.

Although the size of the foci showed a significant difference betweenthe two groups, the focal scores based on human diagnostic criteria didnot differ. This could be due to species differences or more subtledifferences between human SS and the NOD mouse model. A furtherpossibility is that the time of onset of GTP treatment (9 weeks of age)might be relatively late with respect to the initial phases of theprocess of gland damage. NOD-scid congenic mice (that lack functionallymphocytes) do not develop sialadenitis (or diabetes). However, they doshow dysfunction in expression of biochemical markers of salivary glanddifferentiation such as amylase and parotid secretory protein (PSP).These data are consistent with a model for SS in which there is aninitial phase, during which dysregulation of glandular homeostasistriggers the disease, followed by an immune cell-mediated phase thatleads to a loss of secretory function (Cha et al., 2002).

Still another embodiment provides a method for reducing or inhibitingautoimmune destruction of cells expressing an autoantigen by contactingthe cells with an amount of green tea polyphenols, for example ECGC,effective to activate the p38 pathway in the cells.

The multiple MAPK signal transduction pathways are involved in thecontrol of diverse cellular events including proliferation,differentiation and apoptosis. Gene expression in salivary epithelialcells is regulated, in part, via the Raf/MEK/MAPK pathway (Slomiany andSlomiany, 2003, Li et al., 1997). It was found that Raf-1 kinase-induceddown-regulation of a sodium channel was blocked by the MEK inhibitorPD98059, suggesting that the ERK pathway is involved in the signaltransduction (Zentner et al., 1998). The acinar cells respond to nitricoxide (NO), an inflammation-related signaling molecule, by the pathwaysregulated by ERK and p38 (Slomiany and Slomiany, 2002a). The p38 MAPKpathway is important in transducing stress signals, and p38 MAPK isstrongly and rapidly activated by stresses and inflammatory cytokines(Dent et al., 2003). Recently, it was suggested that inhibition of LPS-stimulated iNOS and COX-2 expression and reduced NO release were by amechanism involving p38 (Brautigam et al., 2005). SS patients showactivated forms of p38 and JNK in infiltrating mononuclear cells(Nakamura et al., 1999). Protein kinases downstream of p38 can activatetranscription factors such as activating transcription factor-2 (ATF-2)and growth and DNA damage (GADD)-153 transcription factor. The p38 MAPKfamily consists of at least 4 isoforms. The specificity of the isoformsactivated depends on the cell type, and the nature and strength of thesignals (Morin and Huot, 2004). Importantly, the cellular response top38 MAPK activation is highly cell type dependent: it can induceapoptosis, growth arrest, or differentiation (Slomiany and Slomiany,2002, Dent et al., 2003, Morin and Huot, 2004).

It has been discovered that EGCG induced the activation of p38 byphosphorylation in a dose dependent manner. p38 is rapidly andspecifically phosphorylated within 30 min in acinar cell-derivedNS-SV-AC cells by EGCG, while levels pJNK and pERK were relativelystable (FIG. 8). Further, when NS-SV-AC cells were pre-treated with aspecific inhibitor of p38, EGCG failed to protect these cells fromTNF-α-induced cytotoxicity (FIG. 7A). An inhibitor of MEK, a MAPKupstream of p38, also blocked EGCG protection (FIG. 7B). Taken together,these results implicate the p38/MAPK pathway in mediation of some of thebeneficial effects of GTPs on cell-based mechanisms of disease inSS-affected salivary glands. Accordingly, another embodiment provides amethod for treating an autoimmune disorder by administering to a host anactivator of the p38/MAPK pathway, in particular a green tea polyphenolcompound.

Still another embodiment provides a method for treating xerostomia byadministering to a host one or more green tea polyphenols in an amounteffective to decrease expression of one or more autoantigens in one ormore salivary gland cells of the host. Reducing the expression of anautoantigen in the salivary gland cell can reduce the autoimmunedestruction of the salivary gland cell.

Another embodiment provides a method for treating xerophthalmia byadministering to a host one or more green tea polyphenols in an amounteffective to decrease expression of one or more autoantigens in one ormore of the host's lacrimal gland cells. Reducing the expression of anautoantigen can prevent destruction of lacrimal gland cells.

Still another embodiment provides a method for treating psoriasis byadministering to a host one or more green tea polyphenols in an amounteffective to decrease expression of one or more autoantigens in one ormore epidermal cells of the host. Reducing the expression of anautoantigen can prevent autoimmune destruction of epidermal cells.

Another embodiment provides use of a green tea polyphenol in themanufacture of a medicament for the treatment of an autoimmune disease,for example SS, psoriasis, xerophthalmia, or xerophthalmia.

4. Combination Therapy

The disclosed green tea polyphenol compositions can be used incombination or alternation with one or more additional therapies.Representative additional therapies that can be used with the disclosedcompositions include, but are not limited to non-steroidalanti-inflammatory drugs, antimalarial drugs, corticosteroids,immunosuppressants, antioxidants, antibodies against T-cells orTNF-[alpha], and combinations thereof. Suitable non-steroidalanti-inflammatory drugs include ibuprofen, naproxen, indomethacin,celecoxib or other COX-2 inhibitors, rofecoxib, and combinationsthereof. Treatment for psoriasis includes PUVA (UV light pluspsorialin). Exemplary antimalarials include hydroxychloroquine.Representative corticosteroids include prednisone, prednisolone, andcombinations thereof. Suitable immunosuppressants include azathioprine,methotrexate, cyclosporine, cyclophosphamide, leflunomide,mycophenolate, and combinations thereof. Additionally, herbal extracts,nutraceuticals, vitamins or combinations thereof can be used with or inaddition to the disclosed compositions.

5. Pharmaceutical Compositions

Another embodiment provides pharmaceutical compositions and dosage formswhich include a pharmaceutically acceptable salt of one or more greentea polyphenols, in particular, (-)-epigallocatechin-3-gallate or apharmaceutically acceptable polymorph, solvate, hydrate, dehydrate,co-crystal, anhydrous, or amorphous form thereof. Specific salts ofdisclosed compounds include, but are not limited to, sodium, lithium,potassium salts, and hydrates thereof.

Pharmaceutical unit dosage forms of green tea polyphenols are suitablefor oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal),parenteral (e.g., intramuscular, subcutaneous, intravenous,intraarterial, or bolus injection), topical, or transdermaladministration to a patient. Examples of dosage forms include, but arenot limited to: tablets; caplets; capsules, such as hard gelatincapsules and soft elastic gelatin capsules; cachets; troches; lozenges;dispersions; suppositories; ointments; cataplasms (poultices); pastes;powders; dressings; creams; plasters; solutions; patches; aerosols(e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable fororal or mucosal administration to a patient, including suspensions(e.g., aqueous or non-aqueous liquid suspensions, oil-in-wateremulsions, or water-in-oil liquid emulsions), solutions, and elixirs;liquid dosage forms suitable for parenteral administration to a patient;and sterile solids (e.g., crystalline or amorphous solids) that can bereconstituted to provide liquid dosage forms suitable for parenteraladministration to a patient.

The composition, shape, and type of dosage forms of the green teapolyphenols of the disclosure will typically vary depending on theiruse. A parenteral dosage form may contain smaller amounts of the activeingredient than an oral dosage form used to treat the same disease ordisorder. These and other ways in which specific dosage formsencompassed by this disclosure will vary from one another will bereadily apparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990).

Pharmaceutical compositions and unit dosage forms of the disclosuretypically also include one or more pharmaceutically acceptableexcipients or diluents. Advantages provided by specific compounds of thedisclosure, such as, but not limited to, increased solubility and/orenhanced flow, purity, or stability (e.g., hygroscopicity)characteristics can make them better suited for pharmaceuticalformulation and/or administration to patients than the prior art.Suitable excipients are well known to those skilled in the art ofpharmacy or pharmaceutics, and non-limiting examples of suitableexcipients are provided herein. Whether a particular excipient issuitable for incorporation into a pharmaceutical composition or dosageform depends on a variety of factors well known in the art including,but not limited to, the way in which the dosage form will beadministered to a patient. For example, oral dosage forms such astablets or capsules may contain excipients not suited for use inparenteral dosage forms. The suitability of a particular excipient mayalso depend on the specific active ingredients in the dosage form. Forexample, the decomposition of some active ingredients can be acceleratedby some excipients such as lactose, or when exposed to water. Activeingredients that include primary or secondary amines are particularlysusceptible to such accelerated decomposition.

The disclosure further encompasses pharmaceutical compositions anddosage forms that include one or more compounds that reduce the rate bywhich an active ingredient, for example a green tea polyphenol, willdecompose. Such compounds, which are referred to herein as“stabilizers,” include, but are not limited to, antioxidants such asascorbic acid, pH buffers, or salt buffers. In addition, pharmaceuticalcompositions or dosage forms of the disclosure may contain one or moresolubility modulators, such as sodium chloride, sodium sulfate, sodiumor potassium phosphate or organic acids. A specific solubility modulatoris tartaric acid.

Like the amounts and types of excipients, the amounts and specific typeof green tea polyphenol in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. However, typical dosage forms of the compounds of thedisclosure include a pharmaceutically acceptable salt, or apharmaceutically acceptable polymorph, solvate, hydrate, dehydrate,co-crystal, anhydrous, or amorphous form thereof, in an amount of fromabout 10 mg to about 1000 mg, preferably in an amount of from about 25mg to about 750 mg, more preferably in an amount of from 50 mg to 500mg, even more preferably in an amount of from about 30 mg to about 100mg.

Additionally, the compounds and/or compositions can be delivered usinglipid- or polymer-based nanoparticles. For example, the nanoparticlescan be designed to improve the pharmacological and therapeuticproperties of drugs administered parenterally (Allen, T. M., Cullis, P.R. Drug delivery systems: entering the mainstream. Science. b 303(5665):1818-22 (2004)).

Topical dosage forms of the disclosure include, but are not limited to,creams, lotions, ointments, gels, sprays, aerosols, solutions,emulsions, and other forms know to one of skill in the art. See, e.g.,Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton,Pa. (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed.,Lea & Febiger, Philadelphia, Pa. (1985). For non-sprayable topicaldosage forms, viscous to semi-solid or solid forms including a carrieror one or more excipients compatible with topical application and havinga dynamic viscosity preferably greater than water are typicallyemployed. Suitable formulations include, without limitation, solutions,suspensions, emulsions, creams, ointments, powders, liniments, salves,and the like, which are, if desired, sterilized or mixed with auxiliaryagents (e.g., preservatives, stabilizers, wetting agents, buffers, orsalts) for influencing various properties, such as, for example, osmoticpressure. Other suitable topical dosage forms include sprayable aerosolpreparations wherein the active ingredient, preferably in combinationwith a solid or liquid inert carrier, is packaged in a mixture with apressurized volatile (e.g., a gaseous propellant, such as freon), or ina squeeze bottle. Moisturizers or humectants can also be added topharmaceutical compositions and dosage forms if desired. Examples ofsuch additional ingredients are well known in the art. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Easton,Pa. (1990).

Transdermal and mucosal dosage forms of the compositions of thedisclosure include, but are not limited to, ophthalmic solutions,patches, sprays, aerosols, creams, lotions, suppositories, ointments,gels, solutions, emulsions, suspensions, or other forms known to one ofskill in the art. See, e.g., Remington's Pharmaceutical Sciences, 18thEd., Mack Publishing, Easton, Pa. (1990); and Introduction toPharmaceutical Dosage Forms, 4th Ed., Lea & Febiger, Philadelphia, Pa.(1985). Dosage forms suitable for treating mucosal tissues within theoral cavity can be formulated as mouthwashes, as oral gels, or as buccalpatches. Additional transdermal dosage forms include “reservoir type” or“matrix type” patches, which can be applied to the skin and worn for aspecific period of time to permit the penetration of a desired amount ofactive ingredient.

Examples of transdermal dosage forms and methods of administration thatcan be used to administer the green tea polyphenols of the disclosureinclude, but are not limited to, those disclosed in U.S. Pat. Nos.:4,624,665; 4,655,767; 4,687,481; 4,797,284; 4,810,499; 4,834,978;4,877,618; 4,880,633; 4,917,895; 4,927,687; 4,956,171; 5,035,894;5,091,186; 5,163,899; 5,232,702; 5,234,690; 5,273,755; 5,273,756;5,308,625; 5,356,632; 5,358,715; 5,372,579; 5,421,816; 5,466;465;5,494,680; 5,505,958; 5,554,381; 5,560,922; 5,585,111; 5,656,285;5,667,798; 5,698,217; 5,741,511; 5,747,783; 5,770,219; 5,814,599;5,817,332; 5,833,647; 5,879,322; and 5,906,830, each of which areincorporated herein by reference in their entirety.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal and mucosal dosage formsencompassed by this disclosure are well known to those skilled in thepharmaceutical arts, and depend on the particular tissue or organ towhich a given pharmaceutical composition or dosage form will be applied.With that fact in mind, typical excipients include, but are not limitedto, water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof, to form dosage forms that are non-toxic andpharmaceutically acceptable.

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith pharmaceutically acceptable salts of a green tea polyphenol of thedisclosure. For example, penetration enhancers can be used to assist indelivering the active ingredients to or across the tissue. Suitablepenetration enhancers include, but are not limited to: acetone; variousalcohols such as ethanol, oleyl, an tetrahydrofuryl; alkyl sulfoxidessuch as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide;polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidongrades (Povidone, Polyvidone); urea; and various water-soluble orinsoluble sugar esters such as TWEEN 80 (polysorbate 80) and SPAN 60(sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of the active ingredient(s).Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of theactive ingredient(s) so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different hydrates, dehydrates,co-crystals, solvates, polymorphs, anhydrous, or amorphous forms of thepharmaceutically acceptable salt of a tight junction modulator can beused to further adjust the properties of the resulting composition.

The disclosed green tea polyphenol compositions can also be formulatedas extended or delayed release formulations. Extended and delayedrelease formulations for various active ingredients are known in theart, for example by encapsulation.

6. Encapsulation of Green Tea Polyphenols

In another embodiment, the green tea polyphenols can be incorporatedinto a polymeric component by encapsulation in a microcapsule. Themicrocapsule can be fabricated from a material different from that ofthe bulk of the carrier, coating, or matrix. Suitable microcapsules arethose which are fabricated from a material that undergoes erosion in thehost, or those which are fabricated such that they allow the green teapolyphenol to diffuse out of the microcapsule. Such microcapsules can beused to provide for the controlled release of the encapsulated green teapolyphenol from the microcapsules.

Numerous methods are known for preparing microparticles of anyparticular size range. In the various delivery vehicles of the presentinvention, the microparticle sizes may range from about 0.2 μm up toabout 100 μm. Synthetic methods for gel microparticles, or formicroparticles from molten materials are known, and includepolymerization in emulsion, in sprayed drops, and in separated phases.For solid materials or preformed gels, known methods include wet or drymilling or grinding, pulverization, size separation by air jet, sieve,and the like.

Microparticles can be fabricated from different polymers using a varietyof different methods known to those skilled in the art. Exemplarymethods include those set forth below detailing the preparation ofpolylactic acid and other microparticles. Polylactic acid microparticlesare preferably fabricated using one of three methods: solventevaporation, as described by Mathiowitz, et al. (1990) J. ScanningMicroscopy 4:329; Beck, et al. (1979) Feral. Steril. 31: 545; andBenita, et al. (1984) J. Pharm. Sci. 73: 1721; hot-meltmicroencapsulation, as described by Mathiowitz, et al., ReactivePolymers 6: 275 (1987); and spray drying. Exemplary methods forpreparing microencapsulated bioactive materials are set forth below.

In the solvent evaporation method, the microcapsule polymer is dissolvedin a volatile organic solvent, such as methylene chloride. The green teapolyphenol (either soluble or dispersed as fine particles) is added tothe solution, and the mixture is suspended in an aqueous solution thatcontains a surface active agent such as poly(vinyl alcohol). Theresulting emulsion is stirred until most of the organic solvent hasevaporated, leaving solid microparticles. The solution is loaded withthe green tea polyphenol and suspended in vigorously stirred distilledwater containing poly(vinyl alcohol) (Sigma). After a period ofstirring, the organic solvent evaporates from the polymer, and theresulting microparticles are washed with water and dried overnight in alyophilizer. Microparticles with different sizes (1-1000 μm) andmorphologies can be obtained by this method. This method is useful forrelatively stable polymers like polyesters and polystyrene. Labilepolymers such as polyanhydrides, may degrade during the fabricationprocess due to the presence of water. For these polymers, the followingtwo methods, which are performed in completely anhydrous organicsolvents, are preferably used.

In the hot melt encapsulation method, the polymer is first melted andthen mixed with the solid particles of biologically active material thathave preferably been sieved to less than 50 microns. The mixture issuspended in a non-miscible solvent (like silicon oil) and, withcontinuous stirring, heated to about 5. degree. C. above the meltingpoint of the polymer. Once the emulsion is stabilized, it is cooleduntil the polymer particles solidify. The resulting microparticles arewashed by decantation with a solvent such as petroleum ether to give afree-flowing powder. Microparticles with sizes ranging from about 1 toabout 1000 microns are obtained with this method. The external surfacesof capsules prepared with this technique are usually smooth and dense.This procedure is preferably used to prepare microparticles made ofpolyesters and polyanhydrides.

The solvent removal technique is preferred for polyanhydrides. In thismethod, the green tea polyphenol is dispersed or dissolved in a solutionof the selected polymer in a volatile organic solvent like methylenechloride. This mixture is suspended by stirring in an organic oil (suchas silicon oil) to form an emulsion. Unlike solvent evaporation, thismethod can be used to make microparticles from polymers with highmelting points and different molecular weights. Microparticles thatrange from about 1 to about 300 μm can be obtained by this procedure.The external morphology of spheres produced with this technique ishighly dependent on the type of polymer spray drying, the polymer isdissolved in methylene chloride. A known amount of the green teapolyphenol is suspended or co-dissolved in the polymer solution. Thesolution or the dispersion is then spray-dried. Microparticles rangingbetween about 1 to about 10 μm are obtained with a morphology whichdepends on the type of polymer used.

In one embodiment, the green tea polyphenol is encapsulated inmicrocapsules that comprise a sodium alginate envelope. Microparticlesmade of gel-type polymers, such as alginate, are produced throughtraditional ionic gelation techniques. The polymers are first dissolvedin an aqueous solution, mixed with barium sulfate or some bioactiveagent, and then extruded through a microdroplet forming device, which insome instances employs a flow of nitrogen gas to break off the droplet.A slowly stirred (approximately 100-170 RPM) ionic hardening bath ispositioned below the extruding device to catch the formingmicrodroplets. The microparticles are left to incubate in the bath forabout twenty to thirty minutes in order to allow sufficient time forgelation to occur. Microparticle size is controlled by using varioussize extruders or varying either the nitrogen gas or polymer solutionflow rates.

Liposomes can aid in the delivery of the green tea polyphenol to aparticular tissue and also can increase the half-life of green teapolyphenol. Liposomes are commercially available from a variety ofsuppliers. Alternatively, liposomes can be prepared according to methodsknown to those skilled in the art, for example, as described in Eppsteinet al., U.S. Pat. No. 4,522,811. In general, liposomes are formed fromstandard vesicle-forming lipids, which generally include neutral ornegatively charged phospholipids and a sterol, such as cholesterol. Theselection of lipids is generally guided by consideration of factors suchas the desired liposome size and half-life of the liposomes in the bloodstream. A variety of methods are known for preparing liposomes, forexample as described in Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467(1980); and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369. In one embodiment, the liposomes encapsulating the green teapolyphenol comprise a ligand molecule that can target the liposome to aparticular cell or tissue at or near the site of psoriasis. Ligandswhich bind to receptors prevalent in eplithelial tissue, such asmonoclonal antibodies that bind to epithelial tissue

In one embodiment, the liposomes encapsulating the green tea polyphenolsof the present disclosure are modified so as to avoid clearance by themononuclear macrophage and reticuloendothelial systems, for example byhaving opsonization-inhibition moieties bound to the surface of thestructure. In one embodiment, a liposome can comprise bothopsonization-inhibition moieties and a ligand. Opsonization-inhibitingmoieties for use in preparing the liposomes in one embodiment are largehydrophilic polymers that are bound to the liposome membrane. As usedherein, an opsonization inhibiting moiety is “bound” to a liposomemembrane when it is chemically or physically attached to the membrane,e.g., by the intercalation of a lipid-soluble anchor into the membraneitself, or by binding directly to active groups of membrane lipids.These opsonization-inhibiting hydrophilic polymers form a protectivesurface layer which significantly decreases the uptake of the liposomesby the macrophage-monocyte system (“MMS”) and reticuloendothelial system(“RES”); e.g., as described in U.S. Pat. No. 4,920,016. Liposomesmodified with opsonization-inhibition moieties thus remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation inthe liver and spleen.

Opsonization inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a molecular weight from about 500to about 40,000 daltons, and more preferably from about 2,000 to about20,000 daltons. Such polymers include polyethylene glycol (PEG) orpolypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, andPEG or PPG stearate; synthetic polymers such as polyacrylamide or polyN-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines;polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitolto which carboxylic or amino groups are chemically linked, as well asgangliosides, such as ganglioside GM1. Copolymers of PEG, methoxy PEG,or methoxy PPG, or derivatives thereof, are also suitable. In addition,the opsonization inhibiting polymer can be a block copolymer of PEG andeither a polyamino acid, polysaccharide, polyamidoamine,polyethyleneamine, or polynucleotide. The opsonization inhibitingpolymers can also be natural polysaccharides containing amino acids orcarboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronicacid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid,carrageenan; laminated polysaccharides or oligosaccharides (linear orbranched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups. Preferably, the opsonization-inhibiting moiety is aPEG, PPG, or derivatives thereof. Liposomes modified with PEG orPEG-derivatives are sometimes called “PEGylated liposomes.” Theopsonization inhibiting moiety can be bound to the liposome membrane byany one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH3 and a solvent mixture such as tetrahydrofuran and water in a30:12 ratio at 60° C.

The disclosed microparticles and liposomes and methods of preparingmicroparticles and liposomes are offered by way of example and are notintended to define the scope of microparticles or liposomes of use inthe present disclosure. It will be apparent to those of skill in the artthat an array of microparticles or liposomes, fabricated by differentmethods, are of use in the present invention.

MATERIALS AND METHODS

Chemicals and antibodies: EGCG was purchased from Sigma-Aldrich (St.Louis, Mo.). Anti-human CENP-C (H-300) Rabbit polyclonal antibody,Anti-human 52 KD Ro/SSA (D-12) mouse monoclonal antibody, anti-humanPARP (F-2) mouse monoclonal antibody, anti-human p38, pp38, pJNK andpERK antibodies, and anti-human actin (1-19) goat polyclonal antibodywere purchased from Santa Cruz Biotechnology, Santa Cruz, Calif. Theanti-human coilin mouse monoclonal antibody was obtained from BDTransduction Laboratories, San Jose, Calif. The anti-human Golgin-67rabbit polyclonal antibody was a kind gift from Dr. Don Fujita,University of Calgary, Canada. The anti-human La/SSB mouse monoclonalantibody was purchased from Immunovision Diagnostics Inc., Springdale,Ariz. The mouse Anti-Nuclear Antibodies (ANA) ELISA Kit was purchasedfrom Alpha Diagnostic International, Inc. San Antonio, Tex. Specificinhibitors for p38 (SB 203580), JNK (Sp600125) and ERK (PD 98059) weresupplied by EMD Bioscience, Inc., San Diego, Calif. The 70% GTPs areprovided by Zhejinag Cereals, Oils & Foodstuffs Imp/Exp Co., Ltd, China,which contain 40% EGCG, 13% ECG, 7.3% EGC, 3.2% EC, and 2.7% Caffeine.

Animal treatment: The NOD mice were purchased from Jackson Laboratory.The NOD/LtJ strain develops spontaneous autoimmune symptoms thatresemble human SS (Cha, S. et al. (2002) Crit Rev Oral Biol Med.13:4-16). The NOD mouse is an important model system that has providedclues to the cellular mechanisms involved in SS. Almost 50% of publishedanimal studies for SS used this model during the past two years. Thismouse strain develops a lymphocytic infiltration of exocrine tissues at10-12 weeks of age, particularly in females, and was originally used asa model for type I diabetes. NOD-scid congenic mice (that lackfunctional lymphocytes) do not develop sialadenitis (or diabetes).However, they do show dysfunction in expression of biochemical markersof salivary gland differentiation such as amylase and parotid secretoryprotein (PSP). These data are consistent with a model of SS in whichthere is an initial phase during which dysregulation of glandularhomeostasis triggers the disease, followed by an immune cell-mediatedphase that leads to a loss of secretory function. All animal protocolsin this study were approved by the Institutional Animal Care and UseCommittee. Animals (two groups, 15 NOD mice/group) were allowed adlibitum access to either water or 0.2% GTPs starting at the 9^(th) weekof age. After the onset of autoimmune disease (determined as diabetes,detected by Glucotest strips), each animal was allowed to progress withthe disease for three weeks, and then euthanized (2 water-fed controlmice died during this 3-week period). Blood was collected from eachanimal in the above described treatment groups by cardiac punctureimmediately after euthanasia, and serum for ELISA assays was prepared bycentrifugation of blood samples at 3000 rpm. The submandibular glandswere dissected free of the sublingual gland and attached tissues forpathological analyses.

Determination of serum total autoantibodies: Samples from the two groupswere examined by ELISA assays for anti-SS-associated autoantibodiesusing the Mouse Anti-Nuclear Antibodies (ANA) ELISA Kit (Cat #5200,Alpha Diagnostic International, Inc. San Antonio, Tex.) according to themanufacturer's instructions. This kit detects total ANA against ds-DNA,ss-DNA, histones, ribonucleoproteins (RNPs), SS-A, SS-B, SM antigens,Jo-1, and Scl-70. Samples were analyzed with blanks, positive andnegative controls in 96-well plates by ELISA reaction, photo-detectionusing a VERSAmax Microplate Reader at 450 nm, and statistical analysisusing two-tailed student t-test.

Immunohistochemistry: The submandibular glands from NOD mice were fixedin 10% neutral-buffered formalin, paraffin embedded, sectioned at 5[mu]m, and stained with H&E by routine methods previously described (Hsuet al., 2005).

Pathology scoring of lymphocyte infiltration in the submandibular glandsof NOD mice. We adopted the cumulative focus score (cFS) criteriarecently published by Morbini et al. (101) for the assessment ofsalivary gland inflammatory infiltrates as a component of the diagnosisof Sjögren's syndrome (SS). Briefly, these criteria modify theAmerican-European Consensus Group (Vitali et al., 2002) focus score (FS)criterion as a component of the diagnosis of SS by adopting a multilevelsectioning and evaluation of salivary gland tissues of suspected SSpatients. This multilevel sampling improved the diagnostic accuracy ofbiopsies with a baseline FS between 1 and 2, which represents thecritical cutoff in SS histopathological evaluation (Morbini et al.,2005). The cFS method assesses the number of chronic (lymphocytic)inflammatory cell infiltrates of at least 50 in a 10-HP (equivalent to 4mm²) light microscopy field repeated for a minimum of three differenttissue section levels. The arithmetic average FS from all examinedlevels from a particular gland represents the cFS average for thatgland. To examine differences in the size of foci above 50 cells, aquantitative analysis was performed using the BIOQUANT NOVA PRIME 6.75software. One H&E-stained submandibular salivary gland section wasselected at random for each of the animals in the two groups and imagesof areas containing foci were loaded into the software. The areas oflymphocyte infiltration foci were captured individually and measuredquantitatively by the soft ware as relative density units.

Cell lines: NHEK were purchased from Cambrex (East Rutherford, N.J.) andsub-cultured in KGM-2 provided by the manufacturer. Subculture of theNHEK was performed by detaching the cells in 0.25% trypsin, andtransferring into new tissue culture flasks. The immortalized humansalivary gland acinar cells NS-SV-AC have been previously described(Azuma M. et al. (1993) Lab Invest. 69 (1):24-42). These cells wereselected following transfection of origin-defective SV40 mutant DNA andmaintained in KGM-2 medium, they were kindly provided by Dr. MasayukiAzuma (Tokushima University School of Dentistry, Tokushima, Japan).Subculture of the cells was performed by detaching the cells in 2.5%trypsin, and transferring into new tissue culture flasks.

Cell treatment: EGCG was dissolved in cell culture media as a 50 mMstock immediately before use. Exponentially growing NS-SV-AC cells wereincubated with 100 μM EGCG for various time periods in the growth mediadescribed above, and then were either extracted for total RNA or celllysates prior to RT-PCR or Western blots.

Affymetrix gene array analyses. The gene array experiment was performedat the Core Facility of Genomics of the Medical College of Georgia,according to the instructions of the manufacturer (Affymetrix, SantaClara, Calif.). Briefly, NHEK and OSC2 cells treated with 100 uM EGCGfor different time period were extracted for total RNA, the RNA wasprocessed for probes and hybridized with a cDNA microarray (HuG133A,Affymetrix) which represents 22,283 human series. DNA chips were readwith a Hewlett-Packard GeneArray Scanner at a resolution of 3 mm andwere analyzed with the GeneSpring software (Silicon Genetics, RedwoodCity, Calif). Details of this analysis will be published elsewhere (Hsuet al., manuscript in preparation).

Total RNA extraction and semi-quantitative reverse-transcription PCR(KT-PCR. Total RNA was extracted using an RNeasy total RNA isolationsystem (QIAGEN, Valentia, Calif.) RT reactions and PCR reactions wereperformed according to the manufacturer's protocol (Super ArrayBioscience Corp). For amplification, the following pair of primers wasused, GAPDH: Sense, 5′-TCCCATCACCATCTTCCA-3′ (SEQ ID NO. 1), Antisense,5′-CATCACGCCACACGAGTTTCC-3′ (SEQ ID NO: 2), SS-A/Ro (469 bp): sense5′-GAACTGCTGCAGGAGGTGATAA-3′ (SEQ ID NO: 3), Antisense5′-GGCACATTCAGAGAAGGAGT-3′ (SEQ ID NO: 4), SS-B/La (95 bp): sense5′-CCAAAATCTGTCATCAAATTGAGTATT-3′ (SEQ ID NO: 5), Antisense5′-CCAGCCTTCATCCAGTTTTATCT-3′ (SEQ ID NO: 6), Amplification was startedby heating for 1.5 min at 94° C., followed by 30 cycles for 52 kDSSA/Ro. 25 cycles for GAPDH and 25 cycles for SSB/La, each cycleconsisting of 15 second at 94° C., 30 seconds at 57.3° C., and 1 min at72° C. A final extension was performed at 72° C. for 5 min prior to gelanalysis.

Western blot: Cells were washed in ice-cold PBS and lysed for 20 min inRIPA buffer containing 1% (v/v) Nonidet P-40, 0.5% (w/v) sodiumdeoxycholate, 0.1% (w/v) SDS, 10 μg/ml leupeptin, 3 μg/ml aprotinin and100 mM phenylmethylsulfonyl fluoride (PMSF). Samples of lysatescontaining the same amount of protein were loaded in each lane (we used5-30 μg, depending on the antibody used) and electrophoreticallyseparated on a 12% SDS polyacrylamide gel. Following electrophoresis,proteins were transferred to a PVDF membrane (Immobilon™-P, MilliporeCorporation, Bedford, Mass.). The membrane was blocked for 1 h with 5%(w/v) non-fat dry milk powder in PBST (0.1% Tween-20 in PBS) and thenincubated for 1 h with primary antibody diluted in PBST/milk (Antibodiesand dilutions: rabbit polyclonal JNK1/2, 1:000, mouse monoclonalpJNK1/2, 1:1000, rabbit polyclonal ERK, 1:2000, rabbit polyclonal p38,1:1000, rabbit polyclonal pp38, 1:1000 and goat polyclonal actin,1:2000). The membrane was washed three times with PBST and incubatedwith peroxidase-conjugated, affinity-purified anti-rabbit IgG (SantaCruz Biotechnology, Inc.) for 1 h. Following extensive washing, thereaction was developed by enhanced chemiluminescent staining using ECLWestern blotting detection reagents (Amersham Pharmacia Biotech Inc.,Piscataway, N.J.).

TNF-a cytotoxicity: NS-SV-AC cells (0.5×10<4>cells/well) were seeded ina 96-well microplate and either treated for 24 hr with EGCG as describedabove (or not treated in the control), in the presence of 100 ng/ml TNFαand 10 μg/ml cyclohexamide. After the treatments, viability wasdetermined by the MTT assay. The cells in each well were washed with 200μl of phosphate-buffered saline (PBS) and incubated with 100 μl of 2%(w/v) MTT in a solution of 0.05 M Tris-HCl (pH 7.6), 0.5 mM MgCl₂, 2.5mM CoCl₂ and 0.25 M disodium succinate at 37° C. for 30 min. Cells werefixed by the addition of 100 μl of 4% (v/v) formalin in 0.2 M Tris-HCl(pH 7.6), and after a 5 min incubation at room temperature liquid wasremoved and the wells were allowed to dry. Each well was rinsed with 200μl water and cells were solubilized by the addition of 100 μl of 6.35%(v/v) 0.1 N NaOH in DMSO. The colored formazan product was measured by aThermo MAX micro plate reader (Molecular Devices Corp. Sunnyvale,Calif.) at a wavelength of 562 nm.

EXAMPLES Example 1 Affymetrix Gene Array Analyses

Gene assays provide a valuable tool for studying the broad response ofcells to agents, and we have used this approach to compare thedifferential effects of GTPP on gene expression in NHEK and human oralsquamous carcinoma cells (Hsu, et al., manuscript in preparation). Theresults demonstrated that approximately 2100 genes were either up- ordown-regulated by at least 2-fold in response to 100 uM EGCG in bothcell types during the 24 hrs post-treatment. Examination of the relativeNHEK mRNA levels of genes represented on the chip encoding autoantigenspreviously identified in SLE (Sherer et al., 2004) revealed a 2- fold orgreater change in expression of a number of genes during the 24 hrpost-treatment time course (Table 1). See also Hsu, S. et al. (2005)JPET 315:805-811, which is incorporated by reference in its entirety.

Several patterns of change were observed. For example, the nuclearautoantigen genes (RNA polymerase I and SS-B/La), a cytoskeletalautoantigen gene (alpha-fodrin) and a Golgi autoantigen gene (golgin-67)showed a general pattern of rapid (0.5-2 hr initiation) and persistent2-fold or greater decrease in mRNA levels during the 24 hr period. Othergenes showed an initial decrease during the first 2 hrs (e.g.,centromere protein Cl, coilin), with levels returning to near normal by24 hrs. Other autoantigen genes (e.g., nuclear antigen SP100 andscleroderma autoantigen I) showed a rapid initial increase (2-4-fold)followed by a 2-fold or greater decrease. Other genes (e.g., one of thethee arrayed Ku autoantigen genes) showed a transient initial increase,with levels returning to near normal. In contrast, other antoantigengenes did not show a significant change during the time course of theexperiment. For example, 60 kDa SS-A/Ro did not decline significantly(52 kDa SS-A/Ro cDNA was not included in the gene array).

An additional mechanism by which EGCG modulation of gene expressioncould afford protection in autoimmune disorders might be by reduction inthe expression of proinflammatory signaling molecules. Data show thatthere is a broad reduction in expression of pro-inflammatory genes.Table 3 shows the results for a number of important inflammation-relatedgenes whose expression in NHEK is modulated by EGCG (the entire group istoo large to display). These data show that EGCG can suppress theexpression of many inflammatory factors. Importantly, the MAPK familymember p38 is induced.

TABLE 2 Summary of gene array analysis of autoantigen gene expression.Analysis of the gene array dataset for EGCG-treated normal humanepidermal keratinocytes (NHEK) cells demonstrates a general pattern ofreduction in autoantigen gene expression. Time 0, Time 0.5, Time 02,Time 06, Times 24, Affymetrix Cell Type Cell Type Cell Type Cell TypeCell Type cDNA Array NHEK NHEK NHEK NHEK NHEK Genes coding for Symbolsnormalized normalized normalized normalized normalized autoantigens20473_at 1 1.27 0.46 0.87 0.94 centromere protein C1 203653_s_at 1 0.490.54 0.60 0.79 coilin 203654_s_at 1 1.11 0.78 0.62 0.79 coilin208797_s_at 1 1.26 0.71 0.92 0.29 golgin-67 208798_x_at 1 0.81 0.46 0.320.31 golgin-67 210425_x_at 1 0.50 0.47 0.19 0.31 golgin-67 213650_at 10.24 0.61 0.69 0.72 golgin-67 200792_at 1 1.15 1.19 1.13 0.79 thyroidautoantigen 70 kDa (Ku antigen) 208642_s_at 1 1.29 1.14 0.96 0.73 Kuautoantigen, 80 kDa 208643_s_at 1 2.34 1.40 0.98 0.77 Ku autoantigen, 80kDa 202692_s-at 1 0.50 0.48 0.50 0.55 RNA polymerase 1 208611_s_at 10.41 0.75 0.56 0.42 fodrin-α 212071_s_at 1 0.91 0.92 0.95 0.72spectrin-β non- erythrocyctic 1 215235_s_at 1 0.49 0.97 0.75 0.71fodrin-α 212852_s_at 1 0.60 0.98 0.85 0.94 60 kDa, SS- A/Ro 210438_x_at1 0.71 0.92 0.69 0.88 60 kDa, SS- A/Ro 201139_s_at 1 0.71 0.74 0.43 0.26SS-B/La 201138_s_at 1 0.84 0.88 0.57 0.27 SS-B/La 202863_at 1 1.72 1.040.59 0.36 Nuclear antigen SP100 202864_s_at 1 3.12 1.21 0.64 0.08Nuclear antigen SP100 213226_at 1 3.99 1.09 0.43 0.50 Nuclear antigenSP10075 kDa 213226_at 1 3.99 1.09 0.43 0.50 scleroderma autoantigen 1,75 kDa

TABLE 3 Selected inflammatory gene expression detected from NHEK treatedwith 100 μM EGCG for indicated hours. Time 0, Cell Time 0.5, Cell Time02, Cell Time 06, Cell Times 24, Cell Affymetrix Common Type NHEK TypeNHEK Type NHEK Type NHEK Type NHEK symbols Names normalized normalizednormalized normalized normalized 205067_at IL1B 1 0.29 0.58 0.49 0.2639402_at IL1B 1 0.29 0.48 0.40 0.27 210118_s_at IL1A 1 0.66 0.42 0.540.27 205290_s_at BMP2 1 0.32 0.96 0.72 0.44 206295_at IL18 1 0.33 0.650.70 0.65 206172_at IL13RA2 1 0.33 0.74 0.81 0.55 21100_s_at IL6ST 10.34 0.28 0.99 0.61 209575_at IL10RB 1 0.43 0.63 0.76 0.38 205945_atIL6R 1 0.48 0.69 0.31 0.69 221085_at TNFSF15 1 0.52 1.56 0.67 0.63202727_s_at IFNGR1 1 0.79 0.91 0.45 0.92 202859_x_at IL8 1 6.02 9.559.99 9.93 21156_x_at p38 1 2.32 1.46 1.62 2.51 202530_at p38 1 2.51 1.090.63 1.17 Except p38 and IL8, all other genes were inhibited by EGCG.Numbers represent fold up/down at the time points indicated.

Example 2 Semi-Quantitative RT-PCR

The preliminary array analysis described above provided data suggestingthat GTPP could alter the expression of some genes encodingautoantigens. To further test this possibility, three cell types, NHEK,NS-SV-AC (an SV40 immortalized cell line derived from humansubmandibular acinar cells) and OSC2 cells (derived from an oralsquamous cell carcinoma) were treated with 100 uM EGCG for differenttimes. A semi-quantitative estimate of SS-A/Ro and SS-B/La mRNA levelswas then obtained by RT-PCR during the exponential period ofamplification. GAPDH was used as a housekeeping gene control. SS-B/Laand SS-A/Ro 52 were selected because elevated mRNA levels (2-3 fold) ofSS-A/R0 52 and SS-B/La are found in salivary tissues of SS patients(Bolstad A. I., et al. (2003) Arthritis Rheum 48:174-185), andautoantibodies against SS-A/Ro end SS-B/La are found in nearly all(about 95% and 87%, respectively) primary SS patients (Hahn: 1998).Consistent with the gene array data, SS-R/La message decreasedprogressively and substantially in both NHEK and NS-SV-AC cells (FIG.1). The levels of SS-A/Ro (52 kd) mRNA also showed a reduction duringtreatment, although the effect was less pronounced than that seen forSS-B/La and was not prominent until 24 hrs. In OSC2 cells the reductionin SS-B/La mRNA was less pronounced and did not occur until 24 hrs ofexposure, while SS-A/Ro mRNA showed little change. GAPDII also showed nomarked decrease in these cells in response to EGCG, indicating thereduction in mRNA levels seen for SS-B/La and SS-A/Ro was not due to ageneralized effect on the cells.

Example 3 Protein Levels of Autoantigens

To extend the mRNA data, the protein levels of 6 different autoantigensin NHEK and NS-SV-AC cells were determined by Western analysis followingtwo treatments with 100 uM EGCG at 24 and 48 hrs. As shown in FIG. 2,coilin and PARP protein levels were significantly reduced in both celllines by 24 hrs, and were barely detectable after 48 hrs. CENP-C showeda similar trend, although the reduction was less pronounced. NeitherSS-A/Ro 52 nor SS-B/La were significantly reduced in either cell line by24 hrs, but were considerably reduced by 48 hrs. Golgin-67 was reducedin NS-SV-AC cells by 24 hrs, and barely detectable at 48 hrs. Golgin-67was also reduced in NHEK cells, although the reduction was not as markedand was only observed at 48 hrs. Actin protein levels were unchanged byEGCG during the 48 hr treatment period.

Example 4 Serum Total Autoantibody ELISA

NOD mice were fed either water or water containing 0.2% GTPs for 3weeks. Serum of 27 animals was analyzed: 15 from the GTP-water and 12from the water-only group (two animals died, one animal was not able tocollect serum in the latter group). There was a significant differencebetween the total serum antibody levels from the GTP-water group andwater-only group. On average, the total ANA (against ds-DNA, ss-DNA,histones, ribonucleoproteins [RNPs], SS-A, SS-B, SM antigens, Jo-1, andScl-70) in the GTP-water animal were approximately 20% lower than thatof the water-only animals (FIG. 3). This result indicates that oraladministration of GTPs significantly reduced the serum autoantibodylevels (two-tailed student t-test, p=0.036, n=27).

Example 5 Analysis of Lymphocyte Infiltration

The submandibular glands of each NOD animal were collected and thestandardized scores for the inflammatory cell infiltrates weredetermined blindly, as described in the methods. FIG. 4 showsrepresentative submandibular glands from a water-fed control (A) and aGTP/water-fed NOD mouse (B). Pathological focal scoring, using thecumulative focus score (cFS) criteria for SS diagnosis, demonstrated nosignificant difference in focal scores (i.e. the number of focalinflammatory cell aggregates containing 50 or more lymphocytes in each 4mm² area) between the GTP-treated and untreated (water) controls. Theaverage focal score was 2.125±1.13 for GTP-fed mice and 2.125±0.64 forcontrol mice. However, inspection of the foci suggested potentialdifferences in the focal areas between the groups, equivalent todifferences in the total number of inflammatory cells/focus. Forexample, both animals shown in FIG. 4 received a focal score of 3, butthe foci in the GTP-treated animal appear smaller (although for any onefocal group this might just reflect an off-center cut through itsvolume). Quantitative analysis of the areas of lymphocyte infiltrationfoci in H&E-stained submandibular gland sections (FIG. 5, n=27 animals)demonstrated a statistically significant difference (p=0.006, two-tailedt-test, n=83 foci/group) between the groups in the number ofinflammatory cells/infiltrate, with fewer cells in the salivary glandsof GTPs/water-fed animals.

Example 6 Human Salivary Gland Acinar Cells are Protected fromTNF-α-Induced Cytotoxicity by EGCG

TNF-α, which is produced by inflammatory cells, is known to inducecytotoxicity in many cell types, and can be down-regulated by EGCG(Suganuma et al., 2000, Fujiki et al., 2000, Fujiki et al., 2003).Therefore, one mechanism by which EGCG could ameliorate the effects ofSS could be attenuation of TNF-α-cytotoxicity. We examined the effectsof EGCG on TNF-α-induced cytotoxicity of the human salivary gland acinarcell line NS-SV-AC using the MTT assay. Results from these experimentsare summarized in FIG. 6. EGCG in a dose-dependent manner providedsignificant protection of NS-SV-AC cells (up to 50%) from TNFα-inducedcytotoxicity (two-tailed t-test, p<0.05).

Example 7 38 MAPK Inhibitor SB203580 and MEK Inhibitor PD98059 Abolishedthe Protective Effect of EGCG

To examine the role of activation of the p38 signaling pathway inreducing TNF-α-induced cytotoxicity, cells were exposed to TNF-[alpha]and EGCG in the presence of either the p38 MAPK inhibitor SB203580 orthe MEK inhibitor PD98059. The results of MTT assays indicated that bothinhibitors significantly reduced the protective effect of EGCG againstTNF-α-induced loss of cell viability (FIG. 7).

Example 8 EGCG Specifically Activates the Phosphorylation of p38 inNS-SV-AC Cells

p38 activation is one mechanism by which GTPs attenuates mechanisms ofSS pathogenesis. As shown in FIG. 8, EGCG induced a rapid and sustainedphosphorylation of p38 in NS-SV-AC cells while, only a slight increasein pERK was detected, and phosphorylation of .INK was not altered.Therefore, of these three signaling pathways, EGCG activates primarilyp38 in NS-SV-AC cells.

In the NOD mouse model of SS, oral administration of GTPs lowered thetotal serum autoantibody level and reduced the magnitude of salivarylymphocyte infiltration. In vitro, EGCG protected acinar-derived cellsfrom TNF-α-induced cytotoxicity. These protective effects of GTPs wereassociated with the p38 MAPK-signaling pathway.

Example 9 Pathology Scoring of Lymphocyte Infiltration in theSubmandibular Glands of Early GTP-treated MRL and NOD Mice

FIG. 9 shows representative sections of H&E submandibular glands fromeach experimental group (disease free, diseased animals treated withGTP, and diseased untreated animals) in both strains, together with thecorresponding inflammatory infiltrate score. As shown in FIG. 9, glandsfrom disease-free animals showed cFS of 0; disease plus GTPs a cFS of 1and 2; and disease a cFS of 2.5 and 3, for NOD and MRL, respectively.These data show a marked in vivo effect of GTPs on the progression andseverity of SS-like pathology. Both MRL and NOD mice showed a reductionin focal score, suggesting GTPs effects are not strain specific.

TABLE 1 cDNA microarray determination of autoantigen expression in NHEKtreated with 100 μM EGCG for the indicated hours. 0 0.6 2 6 24Autoantigen h Descriptions Inhibited > 2-fold AKAP12 1 0.808 0.891 0.40.188 A kinase IPREA1 anchor protein igmarion 12 α-NAC 1 0.082 0.5210.0143 0.189 Homo sapiens α-NAC gene for nascent polypeptide- associatedcomplex component CENPB 1 0.803 0.773 0.4

8 0.721 Centomero protein B CENPO3 1 1.260 0.457 0.880 0.021 Centomeroprotein C1 FL131657 1 0.332 0.003 0.435 0.009 Hypothetical proteinFL131657 FLNB 1 0.78 0.86 0.875 0.2

Filamin B, γ (actin-binding protein 278) FLNB 1 1.279 1.168 0.751 0.249Filamin B, B (actin-binding protein 378) GOLGA1 1 0.289 1.122 0.08 0.9Golgi autoantigen, golgin subfamily a, 1 GOLGA2 1 0.822 0.012 0.7090.919 Golgi autoantigen, golgin subfamily a, 2 GOLGA3 1 0.833 0.68 0.6170.419 Golgi autoantigen, golgin subfamily a, 3 GOLGA4 1 0.822 0.7480.005 0.001 Golgi autoantigen, golgin subfamily a, 4 HSAC01 1 0.158 0.7

0.

2 0.562 Nucleolar cystoine-rich protein HUMAUNTIC 1 0.618 0.779 0.1140.517 Nucleolar GTPase LMO4 1 0.72 0.807 0.706 1.011 LIM damelts only 4POLR2A 1 0.12 1.29 1.90 1.02 Polysperace (RNA II (DNA-directed)polypeptide A, 220 kDa POLR2A 1 1.08 0.45 0.41 1.17 Polysperace (RNA II(DNA-directed) polypeptide A, 220 kDa POLR2D 1 1.14 0.

1.01 0.29 Polysperace (RNA II (DNA-directed) polypeptide D POLR2E 1 0.450.

0.37 0.39 Polypeptide E, 25 kDa POLR30 1 0.90 0.78 0.43 0.49 Polysperace(RNA III (DNA-directed) (32 kDa) POLR20 1 1.26 1.00 1.05 0.47Polysperace (RNA III (DNA-directed) (32 kDa) SNRPA 1 1.

0.89 1.05 0.22 Small nuclear ribonucleoprotein polypeptide A SP100 11.72 1.087 0.59 0.956 Nuclear antigen Sp100 SPTAN1 1 0.41 0.75 0.53 0.42Spectrin, α,

 1 (

) SPTAN1 1 0.40 0.07 0.75 0.71 Spectrin, α,

 1 (

) SNRPB 1 0.56 0.18 0.78 0.59 Small nuclear ribonucleoproteinpolypeptide B and B1 SNRPB 1 0.02 0.70 0.07 0.42 Small nuclearribonucleoprotein polypeptide B and B1 SNRPB2 1 0.49 0.07 0.50 0.82Small nuclear ribonucleoprotein polypeptide B* SSA2 1 1.009 0.035 0.7391.17

1F1 NIH_M3O_03 77, H. sapiens cDNA clone IMAGE: 4071854 5′, mRNAsequence SSB 1 0.833 0.879 0.57 0.273 Sjogren's syndrome antigen B

SSB 1 0.711 0.729 0.427 0.257 Sjogren's syndrome antigen B

STRNB 1 0.425 0.029 0.823 0.7

9 Striatin, calmodulin binding protein 9 TOP1 1 0.85 1.09 0.34 0.12Topoisomerase (DNA) I TOP1 1 0.63 0.02 0.02 0.42 Topoisomerase (DNA) IURTF 1 0.498 0.484 0.301 0.448 Upstronin binding transcription factor,RNA poly

 I Induced then inhibited > 2-fold PMSOL1 75 kDa 1 8.993 1.09 0.4810.428 Poly

 autoantigen 1 PSMB2 1 2.551 1.151 1.120 0.421

 activator subunit

SNRPD1 1 2.096 1.255 0.058 0.405 Small nuclear ribonucleoprotein D1polypeptide, 18 kDa SP100 1 9.128 1.305 0.093 0.070 Nuclear antigenSp100 Induced then declined to control levels ADPRT 1 9.547 1.201 0.9920.85 ADP-ribosyltransferase [NAD⁺, poly

polymerase] CENPA 1 7.677 1.095 0.672 0.073 Centromere protein A, 17 kDaMCCSSO 1 1.056 1.101 1.618 0.81 Hypothetical protein NOC5560 MCCSSO 17.07 0.882 2.805 0.002 Hypothetical protein NOC5560 SP110 1 3.735 1.7021.205 0.975 SP110 nuclear body protein SP110 1 4.178 1.804 0.007 0.054SP110 nuclear body protein SNRPT0 1 3.59 1.01 2.28 0.51 Small nuclearribonucleoprotein 70-kDa polypeptide (EN

 antigen) XRCC5 1 2.328 1.403 0.577 0.771 X-ray repair complementingdeflective repair in Chinase beveator cells 5

 b

re

, KA

antigen, 30 kDa) Induced > 2-fold ANXA11 1 1.013 1.25 2.141 1.846Annexin A11 CTDBPL 1 2.83 1.12 2.42 2.10 Carboxyl-terminal domain, RNApolymerase II, polypeptide A, small phosphotace-like Charged < 2-foldCALR 1 0.71 0.943 1.482 1.528 Calroticolla CBARA1 1 0.665 0.773 0.4930.721 CHD4 1 0.763 0.811 0.916 0.84 Chromopolmelin helicase DNA-bindingprotein 4 COLITA1 1 1.749 1.102 1.012 0.084 Collagen, type XVII, σ1CTDSP1 1 0.71 1.00 1.25 1.09 Cathoxyl-terminal domain, RNA polymeraseII, polypeptide A, small phophatase 1 CTDSP2 1 1.01 1.21 0.82 1.71Cathoxyl-terminal domain, RNA polymerase II, polypeptide A, smallphophatase 2

21 1 1.842 0.861 0.722 0.06 Contains the 3′ end of a normal gene similarto NY.REPLY antigen DLAT 1 1.54 0.828 1.001 0.761 Dihydroll

dly Soxotyltransferase (

) component of pyruvain dehydrogenesis complex EPPK1 1 0.783 0.880 1.7180.859 Epiplakin I FBL 1 1.54 1.12 1.09 0.89 Creolin kinase II β subunitcomplete cds,

brillin G22F1 1 1.15 1.183 1.18 0.792 Thyroid autoantigen, 70 kDa (Kuantigen) GMEB3 1 0.084 0.782 0.006 0.745 Glucoso

 modulotory element-binding protein 2 GOLGA5 1 1.008 0.028 0.731 1.494Golgi autoantigen, golgin subfamily a δ GOLGB1 1 0.838 0.701 0.56 0.858Golgi autoantigen, golgin subfamily b, macrogolgin with transmembrain

 1 HARS 1 1.27 1.12 0.83 0.33 Hindlydl-IRNA synthetase HARSL 1 1.12 1.060.85 1.00 Hindlydl-IRNA synthetase-like IMP-2 1 1.016 1.280 0.026 0.003Insulin growth

 mRNA, mRNA binding protein 2 LAD1 1 0.788 0.081 0.254 0.863 Elusion

 (LAD) gene, complete cds LMD4 1 0.0 1.190 1.183 0.859 LIM domain only 1NB 1 0.004 0.08 0.180 0.814 Nadeoatenta PDHB 1 1.070 1.11 0.745 0.818

 dehydrogen

PDX1 1 1.103 0.785 0.046 0.520 R3-binding protein PMNC1B 1 0.058 0.8881.558 0.733 Polymyosit

 autoantigen 2, 110 kDa POLR1B 1 0.07 1.00 0.58 1.03 Polymerase (RNA) Ipolypeptide B, 128 kDa POLR1B 1 0.00 0.06 1.01 1.31 Hypothetical proteinB100P880 POLR2B 1 1.00 1.02 0.09 0.74 Polymerase (RNA) II (DNA-directed)polypeptide B, 110 kDa POLR2C 1 1.32 1.00 1.52 1.88 Polymerase (RNA) II(DNA-directed) polypeptide C, 33 kDa POLR2F 1 0.03 1.00 1.17 0.80Polymerase (RNA) II (DNA-directed) polypeptide F POLR2G 1 1.03 1.02 0.740.89 Polymerase (RNA) II (DNA-directed) polypeptide G POLR2H 1 0.85 0.881.03 1.15 Polymerase (RNA) II (DNA-directed) polypeptide H RALY 1 1.5580.780 1.453 0.612 RNA-binding protein

genic, human RMP-associated with lethal swallows SART1 1 1.930 1.1881.102 0.88 Squamous cell

antigen recogniaed by T cells SL

1 1.022 0.380 1.059 1.111 H. sapiens, similar to e

 e-like antigen, clone,

3000 IMAGE:3139311, mRNA, complete cds. SNRPD1 1 0.704 0.678 0.012 0.817Small nuclear ribonucleoprotein D1 polypeptide, 10 kDa SNRPG 1 1.28 1.031.28 1.12 Small nuclear ribonucleoprotein polypeptide G SOX12 1 1.8040.771 1.24 1.882 SRY-box B SP110 1 1.149 1.028 0.7 0.021 SP110 nuclearbody protein SPTBN 1 0.81 0.08 0.88 0.78 Spectrin, B, non

oytis 1 SSA1 1 0.738 0.019 0.087 0.08 Sjogren's syndrome antigen A2191kDa, ribonucleoprotein autoantigen SSA101) SSA2 1 0.008 0.037 0.8711.584 H. sapiens cDNA FL113838

, clone 17

, highly similar to human 80 kDa ribonucleoprotein

 RNA SSA3 1 0.598 0.88 0.83 0.81 H. sapiens cDNA FL113838

, clone 17

, highly similar to human 80 kDa ribonucleoprotein

 RNA SSNA1 1 1.254 1.17 1.038 0.815 Sjogren's syndrome nuclearautoantigen 1 SSSNA1 1 1.837 1.694 1.035 1.283 Sjogren's syndrome

 autoantigen 1 XRCC8 1 1.87 1.141 0.883 0.725 X-ray repair complementingdefective repels in Chilo

 cells &

 basals rejoining; Ku autoantigen, 80kDa) Numbers represent fold up/downat the time points normalized to time zero levels. P

 protein kinase

 human normal peplike

 determining region Y.

indicates data missing or illegible when filed

1-24. (canceled)
 25. A composition comprising:-(-)epigallocatechin-3-gallate, a pharmaceutically acceptable salt orprodrug thereof, in an amount effective to treat xerostomia.
 26. Thecomposition of claim 25, wherein the amount of(-)-epigallocatechin-3-gallate is effective to inhibit expression of oneor more autoantigens in a a salivary gland cell.
 27. The composition ofclaim 26, wherein the one or more autoantigens is selected from thegroup consisting of SS-A, SS-B, fodrin, centromere protein, golgin-67,coilin, and PARP.
 28. The composition of claim 25, wherein thecomposition further comprises (-)-epicatechin, (-)-epigallocatechin,(-)-epicatechin-3-gallate, proanthocyanidins, enantiomers thereof,isomers thereof, combinations thereof, or prodrugs thereof.
 29. Thecomposition of claim 25, wherein the amount of(-)-epigallocatechin-3-gallate is effective to decrease expression of atleast two autoantigens relative to a control. 30-36. (canceled)
 37. Thecomposition of claim 25 in a mucosal dosage form.
 38. The composition ofclaim 37, wherein the mucosal dosage form is a buccal patch.
 39. Thecomposition of claim 37, wherein the mucosal dosage form is a lozenge.40. The composition of claim 25, further comprising an herbal extract.